抗M_2胆碱能受体抗体致心肌损伤作用及其对T淋巴细胞功能的影响
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摘要
研究背景
上世纪九十年代以来,诸多研究者在多种心血管疾病患者的血清中检测到针对M2胆碱能受体细胞外第二环的自身抗体(Autoantibodies Against M2-muscarinic acetylcholine receptor, M2-AA)。进一步的功能学研究发现,该自身抗体具有类似M2受体激动剂卡巴胆碱的作用,对离体培养的心肌细胞产生负性变时效应,且对受体的刺激作用不易脱敏。因此推测,M2-AA长期存在下,可能通过激活M2受体干扰心脏的正常功能,进而导致心脏的病理损害。
本研究室1998年用合成的M2受体细胞外第二环肽段作为抗原长期免疫大鼠,结果观察到在免疫的过程中,伴随着M2-AA滴度的升高和作用时间的延长,心肌组织出现类似扩心病的病理和功能改变；在免疫过程中,心肌细胞线粒体呈现空泡样变性和心肌细胞灶状丢失,并有淋巴细胞浸润。这一结果高度提示循环血液中的M2-AA可能参与了自身免疫介导的心脏疾患的发生。但是,上述研究采用的动物模型为主动免疫模型,主动免疫时注入动物体内的抗原本身也可能导致心肌损伤的发生,因此该研究尚不足以得出M2-AA可以直接导致心肌损伤的结论；另一方面,主动免疫模型所产生的M2-AA远远高于临床患者血清中该抗体的滴度,因而难以用主动免疫模型研究的结果来解释临床病例。因此,建立血清M2-AA浓度类似临床实际情况的被动免疫动物模型,是进一步研究M2-AA病理生理学意义所必需的基本实验手段。
在前述的主动免疫模型中,受损心肌组织有淋巴细胞浸润也是一个不容忽视的病理现象,提示我们可能有某些因素激活了机体的免疫系统。而免疫系统的激活等导致的免疫系统紊乱是高血压、冠心病、扩心病、肥厚性心肌病及风心病等多种疾病恶化的重要原因,已得到国内外专家的公认。已知T淋巴细胞是执行机体免疫应答的主力军,具有直接杀伤靶细胞、辅助或抑制B细胞产生抗体、对特异性抗原和促有丝分裂原的应答反应以及产生细胞因子等多种生物学功能。根据文献报道,在人类和鼠类的T淋巴细胞上都存在M2受体,副交感神经递质ACh可以通过该受体参与免疫调节作用。鉴于M2-AA具有类似M2受体激动剂的作用,因此推测,循环血液中的M2-AA可能会与T淋巴细胞表面的M2受体结合,进而影响T淋巴细胞的活性。但是,M2-AA是否直接参与T淋巴细胞的激活有待进一步确定。如果参与,其参与的方式有必要进行深入研究。
在机体的特异性免疫应答中,CD4+T细胞是主要的效应T细胞,参与免疫应答的各个阶段,并在免疫调节中发挥关键作用。传统观点将CD4+T细胞分为辅助性T细胞1型(Thelper cell 1, Th1)、辅助性T细胞2型(T helper cell 2, Th2)和调节性T细胞(regulatory T cells, Treg)。近年来的研究发现,机体存在一种参与自身免疫和炎症反应的新型CD4+T细胞-辅助性T细胞17型(T helper cell 17, Th17),该细胞通过分泌产生白细胞介素17(Interleukin 17, IL-17,即IL-17A)、IL-17F、IL-21、IL-22、IL-6和肿瘤坏死因子a (Tumor Necrosis Factor a, TNF-a)等,进而发挥其集体动员、募集及活化中性粒细胞的作用。有研究报道,Th17细胞及其特征性细胞因子IL-17可能是自身免疫性心肌损伤的重要因素。如果能特异性观察M2-AA是否直接影响Thl7细胞的功能活动,则有可能进一步提供M2-AA具有调节免疫细胞功能活性的直接证据,为研究M2-AA的病理生理学意义提供新的切入点。
研究目的
(1)建立与临床患者血清中M2-AA滴度相匹配的M2-AA被动免疫大鼠模型,观察该抗体长期存在是否能够影响心脏的结构和功能,以及机体的免疫功能,以探讨M2-AA是否具有直接导致心肌损伤和调控机体免疫功能的作用。
(2)分离培养大鼠T淋巴细胞,观察M2-AA是否直接影响T淋巴细胞的功能活动,以探讨M2-AA是否可激活T淋巴细胞介导的免疫反应,并探讨M2-AA是否具有除心脏以外的作用位点。
(3)分离培养人外周血单个核细胞,刺激诱导其中初始CD4+T细胞发育分化为Th17淋巴细胞,观察M2-AA是否影响Th17淋巴细胞的功能活动。
研究方法
(1)根据大鼠M2受体细胞外第二环163-184位的氨基酸序列合成抗原肽段,建立主动免疫大鼠模型,采用SA-ELISA法监测M2-AA的产生情况。
(2)利用亲和层析法提纯免疫鼠血清中的M2-AA IgGs, SDS-PAGE凝胶电泳鉴定IgGs的纯度,采用BCA法测定蛋白含量,ELISA法鉴定所提纯的M2-AA IgGs亚类。
(3)选择血清M2-AA阴性大鼠建立M2-AA被动免疫模型,利用MS2000生物信号记录分析系统定期监测大鼠心功能的变化情况,并采集心肌组织标本,经相应处理后进行光镜和电镜观察。
(4)分离M2-AA被动免疫大鼠脾脏T淋巴细胞,进行细胞免疫荧光染色,采用流式细胞术测定CD4+/CD8+T淋巴细胞比值。
(5)急性分离培养正常大鼠脾脏T淋巴细胞,进行细胞免疫荧光染色,采用激光扫描共聚焦显微镜观察鉴定。
(6)采用CCK-8法,检测经M2-AA干预后T淋巴细胞的增殖状况,以观察M2-AA对T淋巴细胞增殖功能的影响；利用ELISA检测试剂盒,测定经M2-AA干预后的T淋巴细胞培养上清中IL-4、IL-10、IL-17、IL-21及IL-22含量。
(7)采用Ficoll-Hypaque密度梯度离心法分离人外周血单个核细胞并培养,加入诱导剂与刺激剂促进初始CD4+T淋巴细胞诱导分化为Th17淋巴细胞,进行细胞免疫荧光染色(包括细胞内因子染色),采用激光扫描共聚焦显微镜观察鉴定。
(8)采用CCK-8法,检测经M2-AA干预后Th17淋巴细胞的增殖水平,以观察M2-AA对Th17细胞增殖功能的影响；利用ELISA检测试剂盒,测定经M2-AA干预后的Th17细胞培养上清中IL-17、IL-21及IL-22含量。
研究结果
1.M2-AA的制备、提纯、定性及定量检测结果
1.1 M2抗原肽段主动免疫大鼠模型建立成功
SA-ELISA法测定结果显示：免疫后2周,与对照组相比,M2免疫组血清M2-AA的水平已明显升高,到8周时达峰值(血清M2-AA的OD值：2.93±0.11νs,s.0.21±0.02,P<0.01),之后到10周,M2-AA水平虽有下降趋势,但仍高于同期对照组(2.82±0.33νs.0.20±0.02,P<0.01)(图1-1)。本结果提示主动免疫模型建立成功。
1.2 M2-AA IgGs的提纯、定性、定量及亚类检测结果
采用亲和层析法提纯免疫鼠血清中的IgGs,得到含M2-AA的总IgGs。经SDS-PAGE凝胶电泳鉴定纯度,结果显示在55KDa和25KDa附近可见清晰的两条带,分别代表IgGs的重链和轻链(图1-2A)。
经BCA法测定纯化的IgGs蛋白浓度为2.39mg／mL。
IgGs亚类测定结果显示,所提纯的M2-AA主要为IgG2a亚类(图1-2B)。
2 M2-AA被动免疫大鼠发生心脏损伤
2.1 M2-AA被动免疫大鼠模型建立成功
M2-AA被动免疫后4周,大鼠血清M2-AA含量已明显高于对照组(OD值：0.355±0.03νs.0.103±0.04,P<0.05),之后M2-AA持续保持在一定水平,到24周时仍高于对照组(OD值：0.388±0.04νs.0.098±0.08,P<0.05)(图1-3)。结果表明M2-AA被动免疫大鼠模型建立成功。
2.2 M2-AA被动免疫大鼠的心功能下降
心功能监测中,LVSP与+dp/dtmax是反映心脏收缩功能的参数,LVEDP与-dp/dtmax反映心脏舒张功能。结果显示：被动免疫16周时,M2-AA免疫组LVEDP (KPa)明显升高
(1.53±0.91νs.同期对照组1.38±0.61,P<0.05)(图1-4B),而+dp/dtmax (KPa/sec)则出现降低(302.43±39.1νs.369.18±30.6,P<0.05)(图1-4C)。到24周时,M2-AA组LVSP (KPa)出现降低(9.06±2.20νs.13.92±2.81,P<0.05)(图1-4A),LVEDP增高(2.96±0.52νs.1.29±0.61,P<0.01),+dp/dtmax降低(213.06±40.32νs.360.32±26.81KPa/s,P<0.01),-dp/dtmax(KPa/sec)出现增加(-163.96±28.52 vs.-225.29±12.71 KPa/s, P＜0.01)(图1-4D)。
综上所述,M2-AA长期存在可导致心功能下降。
2.3 M2-AA被动免疫大鼠心重/体重比值下降
心重与体重比值(HW/BW, mg/g)测定结果显示：在免疫第16周时,M2-AA免疫组的HW/BW值明显降低(2.23±0.10νs.对照组2.48±0.11,P<0.05),到24周时降低更为明显(2.15±0.09νs.对照组2.51±0.07,P<0.01)(图1-5C)。这一结果提示,M2-AA在体内长期作用的情况下,可能发生了心肌细胞丢失现象。
2.4 M2-AA被动免疫大鼠导致心肌损伤
心脏体视学检查发现：在免疫24周时,M2-AA免疫组的心脏体积明显变大,心腔扩张(图1-5 A,B)。
光镜观察结合图像分析发现：免疫24周时,M2-AA免疫组心肌细胞形态发生明显改变,间质有淋巴细胞浸润；而对照组心肌组织形态与间质均正常(图1-6)。
观察心肌组织电镜标本发现：免疫24周时,M2-AA免疫组的心肌出现明显的线粒体肿胀、广泛空泡样变性、基质变清、线粒体嵴结构不清；心肌肌原纤维排列紊乱、明暗带缺损、部分断裂。对照组心肌超微结构显示线粒体大小、形态及分布正常；心肌肌原纤维排列整齐,肌小节结构清晰(图1-7)。
以上这些结果说明,M2-AA在体内长期存在的情况下,可导致心肌损伤。
2.5 M2-AA被动免疫后大鼠脾脏CD4+/CD8+T淋巴细胞比值增加
实验结果显示：免疫12周时,M2-AA免疫组的CD4+/CD8+比值明显增高(3.06±0.28νs.对照组1.92±0.38,P<0.05),之后呈持续增高趋势,到24周时仍明显高于对照组(3.47±0.31νs.1.82±0.33,P<0.01)(图1-8)。
这一结果提示,M2-AA在体内长期存在的情况下,可导致机体的细胞免疫功能发生紊乱。
3 M2-AA促进大鼠T淋巴细胞的增殖与分泌作用
3.1成熟T淋巴细胞的激活及鉴定
于倒置显微镜下观察可见：在分离的大鼠脾淋巴细胞中未加入ConA时,淋巴细胞呈圆形；加入ConA后刺激培养72h,细胞的体积增大,数量明显增多,形态有不规则形、长梭形、三角形等(图2-1)。
根据T淋巴细胞特征性的表面标志,采用荧光标记抗体CD3-FITC与CD4-PE进行鉴定。结果可见大部分细胞膜表面呈现绿色荧光阳性,表明是T淋巴细胞；少数细胞膜表面呈绿色与红色荧光双阳性,为辅助性T细胞(Th细胞)(图2-2)。
3.2 Con A可激活大鼠脾脏T淋巴细胞
采用CCK-8试剂盒检测淋巴细胞活性结果显示：体外培养的大鼠脾淋巴细胞中加入ConA后,细胞活性明显高于未加ConA的对照组(0.442±0.05νs.0.126±0.03,P<0.01)(图2-3)。表明Con A能够成功激活T淋巴细胞。3.3 M2-AA浓度依赖性促进大鼠T淋巴细胞增殖
实验结果显示：0.1μmol/L的M2-AA使细胞的活性水平明显升高(OD值：0.561±0.08νs.空白对照组0.439±0.05,P<0.05)(图2-4),同一浓度的氧化震颤素(选择性M2受体激动剂)也具有促T淋巴细胞增殖作用(P<0.05)。
三个浓度的M2-AA(0.01,0.1,1μmol/L)可浓度依赖性促进T淋巴细胞增殖(OD值：0.01：0.496±0.06νs.0.311±0.05,P<0.05；0.1：0.687±0.08νs.0.420±0.06,P<0.01；1：0.783±0.11νs.0.461±0.08,P<0.01)(图2-5)。同样浓度的氧化震颤素也能够浓度依赖性促进T淋巴细胞增殖。3.4 M2-AA通过M2受体介导促T淋巴细胞增殖作用
实验结果显示：T淋巴细胞悬液中同时加入M2-AA与M2受体拮抗剂Methoctraine(美索曲明)的混合液,M2-AA对T淋巴细胞的促增殖作用被抑制(图2-6)。此外,T淋巴细胞悬液中同时加入M2-AA与M2R peptide(M2受体肽段)的混合液,M2-AA对T淋巴细胞的促增殖作用也被抑制(图2-7)。这一结果表明：M2-AA是通过特异性与T淋巴细胞M2受体的细胞外第二环结合,进而实现其促T淋巴细胞增殖作用。
3.5 M2-AA能够促进T淋巴细胞分泌IL-4与IL-10
实验结果显示：0.1μmol/L与1μmol/L的M2-AA可明显增加T淋巴细胞IL-4的分泌量(图2-8)与IL-10的分泌量(图2-9),而对IL-17、IL-21、IL-22的分泌量无明显影响(表1)。
4 M2-AA促进人Th17淋巴细胞的增殖与分泌作用
4.1 Thl7淋巴细胞的形态学观察与鉴定
于倒置显微镜下观察可见：细胞液中未加任何刺激剂时,淋巴细胞呈正常的圆形；而加入一系列的刺激剂培养7天后,细胞形态发生明显改变,有不规则形、长梭形、三角形等,表明细胞已发生明显的分化(图3-1)。
由于目前尚未发现Th17淋巴细胞特征性的表面标志,故采用胞膜CD3-FITC与CD4-APC荧光抗体双染,结合胞内细胞因子IL-17-PE染色的方法进行鉴定。结果细胞膜表面呈绿色与蓝色荧光双阳性,且胞内发红色荧光者即为Thl7淋巴细胞(图3-2)。
4.2 M2-AA浓度依赖性促进Th17淋巴细胞增殖
加入诱导剂与激活剂后,Th17淋巴细胞的活性明显升高(0.398±0.12νs.对照组0.051±0.03,P<0.01)(图3-3)。表明加入诱导剂与激活剂,可使Thl7淋巴细胞活化成熟。
0.1μmol/L的M2-AA使Th17细胞活性水平明显升高(0.554±0.09νs.对照组0.419±0.05,P<0.05)(图3-4)。同一浓度的氧化震颤素也表现出相同的效应(P<0.05)。
三个浓度的M2-AA(0.01,0.1,1μmol/L)可浓度依赖性促进Th17淋巴细胞增殖(0.01：0.443±0.03νs.0.404±0.03,P<0.05；0.1：0.554±0.09νs.0.424±0.06,P<0.01；1：0.569±0.07νs.0.449±0.06,P<0.01)。同样浓度的氧化震颤素也能够浓度依赖性促进Th17淋巴细胞增殖(图3-5)。4.3 M2-AA通过M2受体介导促Th17淋巴细胞增殖作用
Thl7淋巴细胞悬液中同时加入M2-AA与M2受体拮抗剂Methoctraine(美索曲明)的混合液,M2-AA对Th17淋巴细胞的促增殖作用被抑制(图3-6)。此外,Th17淋巴细胞悬液中同时加入M2-AA与M2R peptide(M2受体肽段)的混合液,M2-AA对Th17淋巴细胞的促增殖作用也被抑制(图3-7)。这一结果表明：M2-AA是通过特异性与Th17淋巴细胞M2受体的细胞外第二环结合,进而实现其促Th17淋巴细胞增殖作用。4.4 M2-AA能够促进Th17细胞分泌IL-17、IL-21与IL-22
0.1μmol/L的M2-AA可明显增加Thl7细胞IL-17的分泌量(0.049±0.01νs.0.043±0.01,P<0.05)(图3-8),及IL-21的分泌量(0.048±0.01 vs.0.042±0.01,P<0.05)(图3-9),并且1μmol/L的M2-AA这些作用更为明显(P<0.01)。此外,1μmol/L的M2-AA可明显增加Th17细胞IL-22的分泌量(0.053±0.01νs.0.044±0.01,P<0.05)(图3-10)。同样,0.1μmol/L与1μmol/L的氧化震颤素也可增加Th17细胞对这三种细胞因子的分泌量。结论(1)M2-AA以和临床患者血清滴度相匹配的浓度在体内长期存在的情况下,可引发心肌损
伤和机体细胞免疫功能紊乱。(2)在一定范围内,M2-AA可浓度依赖性促进体外培养的T淋巴细胞增殖,并且增殖的T
淋巴细胞分泌功能增强。表明除心肌细胞以外,T淋巴细胞的M2受体也是M2-AA的
作用靶点。(3)在一定范围内,M2-AA可剂量依赖性促进体外培养的Th17淋巴细胞增殖,并且增殖
的Th17淋巴细胞分泌功能增强。
Background
Since the nineties of last century, many researchers found that there were autoantibodies against the second extracellular loop of M2-muscarinic acetylcholine receptor (M2-AA) in the serum of patients with cardiovascular disease in high titer. Further functional studies found that the M2-AA played a role of negative chronotropic effect on the myocardial cells in vitro, which was similar to M2 receptor agonist carbachol, but the stimulation is not easy to desensitization. Therefore, we speculate that maybe the M2-AA is able to interfere with the nomal function of heart according to activating M2 receptor in the long-term, which result in the pathological injuries to the heart.
We have immunized rats with the synthesis peptides against the second extracellular loop of the M2 receptor as antigens for long time, then we observed:with the increase of M2-AA titer and extend of interaction time, myocardial remodeling occurred gradually, in the end, the the pathological and functional changes which was similar to of dilated cardiomyopathy appeared. During the immunization time, the degeneration of vacuolar presented in mitochondria of myocardial cells, with the loss of myocardial cells in focus, and filtration of lymphocytes. These results suggested that the M2-AA may be involved in autoimmune-mediated pathogenesis of heart disease.However, the above-mentioned study is not sufficient to conclude that M2-AA can result in cardiac injuries directly. Because the model was active immunization model, in one hand, the else factors except M2-AA can not be excluded in the model, for instance, whether the antigen is involved in the occurance of cardiac injuries; on the other hand, the titer of M2-AA produced by active immunization model highly more than the serum of the clinical patients. Thus, it is difficult to explain the clinical cases by active immunization model. Consequently, establishment of passive immunization model of animals whose concentration of M2-AA similar to the clicical situations is the essential and basic experiment method of the further study about the pathophysiology significance of M2-AA.
In the active immunization model above, the infiltration of a large number of lymphocytes in the damaged myocardial tissue was also an important pathological phenomenon, hinting that a certain factors activated the immune system of an organism. As we all know, T lymphocytes are the responsible for the immunologic response, which have multiple biological functions of killing the target cells directly, assisting or inhibiting the antibodies produce by B cells, specific response of antigens and promoting mitogen, and production of cytokines and such on. It is reported that there are M2 receptors in the T lymphocytes of human and murine, and the parasympathetic neurotransmitter ACh plays a role in immune regulation. In view of M2-AA is similar to agonist-like activation to M2 receptors, so we speculated that M2-AA in the circulating blood may also activate M2 receptors of T lymphocytes, thereby affecting the activity of T lymphocytes and immune function. However, whether M2-AA is directly involved in activation of T lymphocytes needs to be further identified. If it is, the forms of participation need to be studied.
In the specific immune response of body, CD4+T lymphocytes are the main effective T cells involved in all stages of the immune response, and play a key role in immune regulation. According to the traditional view, CD4+T lymphocytes were divided into type 1 T helper cells (T helper cell 1, Th1), type 2 T helper cells (T helper cell 2, Th2) and regulatory T cells (regulatory T cells, Treg). In recent years, researchers found that there is a new CD4+T lymphocytes intype17 T helper cells (T helper cell 17, Th17), which involved in body's own immune and inflammatory responses. Th17 lymphocytes can secret interleukin-17 (IL-17, namely IL-17A), IL-17F, IL-21, IL-22, IL-6, TNF-a (Tumor Necrosis Factorα,) and so on. These cytokines play the role of collective mobilization, collection and activated neutrophils. Some studies reported that Th17 cells and their characteristic cytokines IL-17 may be an important autoimmune factors in myocardial injury. If we can confirm whether M2-AA directly affect the functional activity of Th17 cells specifically, may be it will provide direct evidence that M2-AA is capable of regulating immune cell function and activity, which supply a new starting point of further study of the physiological significance to M2-AA.
Objective
(1) The passive immunization rat models were establishment by injection of M2-AA to observe whether long-term presence of M2-AA can lead to changes of cardiac structure and function, as well as in immune function of body, in order to explore whether the M2-AA has a direct role in leading to myocardial injury and regulation of immune function.
(2) Isolation and culture of rat T lymphocytes to observe whether the M2-AA could affect the proliferation and secretion of T lymphocytes, in order to investigate whether the M2-AA can activate T-lymphocyte-mediated immune response, and explore other role sites of M2-AA except to the heart.
(3) Isolation and culture of human peripheral blood mononuclear cells, the native CD4+T cells were induced by the inducers and activators to differentiate to Th17 lymphocytes, in order to observe the influence of M2-AA on functional activites of Th17 lymphocytes.
Methods
(1) The rats model of active immunization are established with synthetic antigenic peptide according to 163-184 amino acid sequence of the second extracellular loop of M2 receptor, and SA-ELISA method is used for monitoring the production of M2-AA.
(2) Affinity chromatography is used to purify M2-AA IgGs in the serum of the immune rats. SDS-PAGE gel electrophoresis is used for determine the purity of IgGs, and BCA method is used for measuring the content of proteins. ELISA method is used for identifying the purified M2-AA IgGs subsets.
(3) Passive immunization rat models of M2-AA were established by the choosing the serum M2-AA negative animal. The MS2000 Biological Signal Recording and Analysis System were used to monitor the cardiac function in a regular time, and myocardial tissue samples were collected and treated to observe under the light and electron microscope.
(4) The T lymphocytes from spleen in rats passive immunization M2-AA were isolated. Cell staining by immunofluorescence was used, and CD4+/CD8+T-lymphocyte ratio is determined by flow cytometry.
(5) The T lymphocytes from spleen of normal rats were acutely isolated and cultured, then the cell staining by immunofluorescence was used and the T lymphocytes were identified by laser scanning confocal microscope.
(6) CCK-8 method was used to detect the activity levels of T-lymphocyte after the accession of M2-AA, in order to determinate the impact of M2-AA on the proliferation function of T-lymphocyte. ELISA kits wer used to detect the levels of IL-4, IL-10, IL-17, IL-21 and IL-22 in the supernatant of cultured T-lymphocyte after intervention by M2-AA.
(7) Ficoll-Hypaque density gradient centrifugation was used to isolate and culture the peripheral blood mononuclear cells in human. Th17 cells were promoted to differentiate and maturate by adding inducing agents and stimulants, and cell staining by immunofluorescence (including intracellular cytokine staining) was used, in order to identify them by laser scanning confocal microscope.
(8) CCK-8 method was used to detect activity level of Th17 cells after intervention by M2-AA, to observe the impact on the proliferation function of Th17 cells by M2-AA; ELISA kits were used to detect the levels of IL-17, IL-21 and IL-22 in the supernatants of cultured Th17 cell after intervention by M2-AA.
Results
1. Preparation, purification, qualitation and quantitation of M2-AA
1.1 The active immunized rat models were set up successfully
The results determined by SA-ELISA showed that 2 weeks after immunization, the level of M2-AA in the sera of M2 immunized group has already raised and reached the peak in 8 weeks (OD Values:2.93±0.11 vs.0.21±0.02, P＜0.01). Then to 10 weeks, the levels of M2-AA decreased, but it was still higher than that before initial immunization (P＜0.01) and the control group (P＜0.01). (Fig.1-1). The result suggests that the active immunized rat models were set up successfully.
1.2 Purification, quantitative qualitative and detection the subtype of M2-AA IgGs
IgGs in the sera of immunized group were purified by affinity chromatography, so the total IgGs including M2-AA were obtained. The specificity of purified IgGs was determined by the SDS-PAGE. The results showed that two straps of 55KD and 25KDa appeared which represented one heavy chain and one light chain, respectively (Fig.l-2A).
The concentration of purified IgGs was 2.39 mg/mL, which was determined by BCA kits.
The subtype of purified M2-AA IgGs was mainly IgG2a (Fig.l-2B).
2. The heart injury appeared in the M2-AA passive immunized rat models
2.1 The passive immunized rat models were established successfully
4 weeks after passive immunization, the sera level of M2-AA in the immunized group was significantly higher than that in the control group (0.355±0.03 vs.0.103±0.04, P<0.05). Then the sera level of M2-AA persisted at stable level. It was still higher than that in control group in 24 weeks after immunization (0.388±0.04 vs.0.098±0.08, P＜0.01) (Fig.1-3). The result suggests the passive immunized rat models were set up successfully.
2.2 The cardiac function decreased in the passive immunized rat
16 weeks after passive immunization, the LVEDP in the M2-AA group has been higher than that in the control group (1.53±0.91 vs.1.38±0.61, P<0.05) (Fig.l-4B), while the+dp/dtmax was significantly lower than the control group (302.43±39.1 vs.369.18±30.6, P＜0.05) (Fig.l-4C). In 24 weeks in the M2-AA immune rats, the LVSP was lower than that in the control group (9.06±2.20 vs.13.92±2.81, P<0.05), but the LVEDP was higher than the corresponding control group (2.96±0.52 vs.1.29±0.61, P<0.01),+dp/dtmax decreased (213.06±40.32 vs.360.32±26.81, P<0.01) (Fig.l-4A), and the-dp/dtmax increased(-163.96±28.52 vs.-225.29±12.71, P<0.01) (Fig.l-4B, D), these results indicate decline in cardiac function.
2.3 The ratios of heart weight to body weight (HW/BW) decreased in the passive immunized rats
16 weeks after passive immunization, the ratio of heart weight to body weight (HW/BW) was significantly lower than that in the control group (2.23±0.10 vs.2.48±0.11, P＜0.05), and it decreased continuously until the experiment was ended up (Fig.1-5C).
2.4 Myocardial injury induced by passive immunization rats with M2-AA
Heart stereological examination showed 24 weeks after immunization with M2-AA, the heart size became larger and the heart chamber expanded (Fig.l-5A, B). While there were no significant changes in the heart at other time points of immunization.
HE staining of myocardial tissue showed:24 weeks after immunization, there were immune inflammatory cells infiltration in the myocardial cells of M2-AA group, and the emergence of infiltration with inflammatory cells, while the control group without such changes in myocardial tissue (Fig.1-6).
Electron microscope displayed marked myocardial fibrosis, deposition with collagen in sarcoplasmic reticulum, and swelling and degeneration in mitochondria, while the control group does not appear similar to myocardial change(Fig.l-7).
These results suggest that long-standing of M2-AA in vivo can lead to myocardial injury.
2.5 The ratios of CD4+/CD8+ T-lymphocytes from spleen cells increased in passive immunization of rats with M2-AA
Before 12 weeks after immunization, there was no significant difference in ratios of CD4+/CD8+ T-lymphocytes in the M2-AA immune group and the control group. It was significantly higher in the M2-AA group at 12th week (3.06±0.28 vs.1.92±0.38, P＜0.05), and it continuous increase to 24 weeks with more notable difference (3.47±0.31 vs.1.82±0.33, P＜0.01) (Fig.1-8).
This result indicates that long-standing of M2-AA in vivo leads to cellular immune function disturbance in the body.
3. M2-AA accelerated proliferation and secretion of T lymphocytes
3.1 The activation and identification of mature T lymphocytes
The T lymphocytes were round or oval-shaped without ConA; when were stimulated and cultured for 72h by, the cell volume augmented, with the number increased significantly, and the pattern were irregular-shaped, long spindle or triangular, etc(Fig.2-1).
The T lymphocytes were identified by immunofluorescence, according to the characteristic surface markers of T lymphocytes. The fluorescently labeled antibodies of CD3-FITC and CD4-PE were used. The result showed most of the cell surface display of green fluorescent-positive, indicating that T lymphocytes; a small number of cell surface was green and red fluorescence double positive, these were the helper T cells (Th cells) (Fig.2-2).
3.2 T lymphocytes from rat spleen could be activated by Con A
After adding ConA to the cultured rat spleen lymphocytes, the cell activity was significantly higher than that of the control group (OD value,0.442±0.05 vs.0.126±0.03, P＜0.01) (Fig.2-3), which indicated that Con A could promote T lymphocytes proliferation.
3.3 M2-AA promoted proliferation of T lymphocytes in a dose-dependent manner
O.1μmol/L of the M2-AA elevated the cell activity significantly (OD values,0.561±0.08 vs. 0.439±0.05, P<0.05), the Oxotremorine also showed the similar effect in the same concentration (P＜0.05) (Fig.2-4).
Three concentrations of M2-AA (0.01,0.1, 1μmol/L) could promote T lymphocyte proliferation in a concentration-dependent manner (OD values:0.01:0.496±0.06 vs.0.311±0.05, P<0.05; 0.1:0.687±0.08 vs.0.420±0.06, P＜00.01; 1:0.783±0.11 vs.0.461±0.08, P＜0.01). The same concentration of Oxotremorine also promoted T lymphocytes proliferation in a dose-dependent (Fig.2-5).
3.4 M2-AA promoted T-lymphocyte proliferation through the M2 receptor
The effect of M2-AA on the proliferation of T lymphocytes could be inhibited by the M2 receptor antagonist Methoctraine (Fig.2-6). In addition, the effect of M2-AA could also be inhibited by M2R peptides (Fig.2-7). The results indicate that the M2-AA promoted T-lymphocyte proliferation through the M2 receptor. 3.5 M2-AA increased the secretion of IL-4 and IL-10 by T-lymphocyte
0.1μmol/L and 1μmol/L of the M2-AA were able to increase the secretion of IL-4 (Fig.2-8) and IL-10 (Fig.2-9) by T-lymphocyte, while M2-AA had no effect on the secretion of IL-17, IL-21or IL-22(Tab.1).
4. M2-AA promoted the proliferation and secretion of Th17 lymphocytes
4.1 The morphology and identification of Th17 lymphocytes
The lymphocytes showed a normal round or oval without any stimulant. When added stimulants for 7 days, the cell morphology changed significantly, they were in irregular shapes, spindle-cell type, triangular, etc. Which indicated that the Thl7 lymphocytes have been differentiated (Fig.3-1).
As there are no characteristic surface markers of Th17 cells so far, we used the membrane CD3-FIT.C and CD4-APC fluorescence antibody staining, combined with intracellular cytokine IL-17-PE staining methods to identify the Th17 lymphocytes. The surface of Th17 lymphocytes cell were green and blue fluorescent double-positive, moreover, the red fluorescence was detected in intracellular (Fig.3-2).
4.2 M2-AA induced a dose-dependent proliferation of Th17 lymphocytes
After added the inducers and activators, the activities of Th17 lymphocytes increased significantly (0.398±0.12 vs.0.051±0.03, P＜0.01), which suggested that add inducers and activators could promote the mature of Th17 lymphocytes (Fig.3-3).
0.1μmol/L of the M2-AA increased the activity of Th17 lymphocytes (0.554±0.09 vs. 0.419±0.05, P<0.05). The same concentration of Oxotremorine also exhibited the same effects (P＜0.05) (Fig.3-4).
Three concentrations of M2-AA (0.01,0.1, 1μmol/L) promoted Th17 lymphocytes proliferation in a concentration-dependent manner (0.01:0.443±0.03 vs.0.404±0.03, P<0.05; 0.1: 0.554±0.09 vs.0.424±0.06, P<0.01; 1:0.569±0.07 vs.0.449±0.06, P＜0.01). The same concentration of Oxotremorine also promoted Th17 lymphocytes proliferation by a concentration-dependent manner (Fig.3-5).
4.3 M2-AA promoted Th17 lymphocyte proliferation through the M2 receptor
The effect of M2-AA on the proliferation of Th17 lymphocytes could be inhibited by the M2 receptor antagonist Methoctraine (0.449±0.03 vs.0.439±0.05, P>0.05) (Fig.3-6). In addition, the effect of M2-AA could also be inhibited by M2R peptides (0.437±0.04 vs.0.439±0.05, P>0.05) (Fig.3-7). These results indicate that the M2-AA promoted Th17 lymphocytes proliferation through the M2 receptor.
4.4 M2-AA promoted Th17 cell secretion of IL-17, IL-21 and IL-22
0.1μmol/L of the M2-AA increased the secretion of IL-17 (0.049±0.01 vs.0.043±0.01, P＜0.05) and IL-21 (0.048±0.01 vs.0.042±0.01, P＜0.05) by the Thl7 lymphocytes (Fig.3-8,3-9), the effects of 1μmol/L M2-AA were more obvious (P＜0.01). In addition, 1μmol/L of the M2-AA increased the secretion of IL-22 (0.053±0.01 vs.0.044±0.01, P＜0.05) (Fig.3-10). Similarly, 0.1μmol/L and 1μmol/L Oxotremorine also increased the secretion of the three kinds of cytokines.
Conclusions
(1) The M2-AA can lead to myocardial injury and cellular immune function disturbance in the condition of long-standing and match the concentration of the clinical serum titer.
(2) Within a certain range, M2-AA promotes T-lymphocyte proliferation and enhances the secretion of cytokines in a concentration-dependent manner in vitro. In addition to the cardiomyocyte, the T lymphocytes are also the role of M2-AA target.
(3) Within a certain range, M2-AA promoted the proliferation of Th17 lymphocytes in dose-dependent manner in vitro, and increased the function of secretion inflammatory cytokines.
上世纪九十年代以来,诸多研究者在多种心血管疾病患者的血清中检测到针对M2胆碱能受体细胞外第二环的自身抗体(Autoantibodies Against M2-muscarinic acetylcholine receptor, M2-AA)。进一步的功能学研究发现,该自身抗体具有类似M2受体激动剂卡巴胆碱的作用,对离体培养的心肌细胞产生负性变时效应,且对受体的刺激作用不易脱敏。因此推测,M2-AA长期存在下,可能通过激活M2受体干扰心脏的正常功能,进而导致心脏的病理损害。
本研究室1998年用合成的M2受体细胞外第二环肽段作为抗原长期免疫大鼠,结果观察到在免疫的过程中,伴随着M2-AA滴度的升高和作用时间的延长,心肌组织出现类似扩心病的病理和功能改变；在免疫过程中,心肌细胞线粒体呈现空泡样变性和心肌细胞灶状丢失,并有淋巴细胞浸润。这一结果高度提示循环血液中的M2-AA可能参与了自身免疫介导的心脏疾患的发生。但是,上述研究采用的动物模型为主动免疫模型,主动免疫时注入动物体内的抗原本身也可能导致心肌损伤的发生,因此该研究尚不足以得出M2-AA可以直接导致心肌损伤的结论；另一方面,主动免疫模型所产生的M2-AA远远高于临床患者血清中该抗体的滴度,因而难以用主动免疫模型研究的结果来解释临床病例。因此,建立血清M2-AA浓度类似临床实际情况的被动免疫动物模型,是进一步研究M2-AA病理生理学意义所必需的基本实验手段。
在前述的主动免疫模型中,受损心肌组织有淋巴细胞浸润也是一个不容忽视的病理现象,提示我们可能有某些因素激活了机体的免疫系统。而免疫系统的激活等导致的免疫系统紊乱是高血压、冠心病、扩心病、肥厚性心肌病及风心病等多种疾病恶化的重要原因,已得到国内外专家的公认。已知T淋巴细胞是执行机体免疫应答的主力军,具有直接杀伤靶细胞、辅助或抑制B细胞产生抗体、对特异性抗原和促有丝分裂原的应答反应以及产生细胞因子等多种生物学功能。根据文献报道,在人类和鼠类的T淋巴细胞上都存在M2受体,副交感神经递质ACh可以通过该受体参与免疫调节作用。鉴于M2-AA具有类似M2受体激动剂的作用,因此推测,循环血液中的M2-AA可能会与T淋巴细胞表面的M2受体结合,进而影响T淋巴细胞的活性。但是,M2-AA是否直接参与T淋巴细胞的激活有待进一步确定。如果参与,其参与的方式有必要进行深入研究。
在机体的特异性免疫应答中,CD4+T细胞是主要的效应T细胞,参与免疫应答的各个阶段,并在免疫调节中发挥关键作用。传统观点将CD4+T细胞分为辅助性T细胞1型(Thelper cell 1, Th1)、辅助性T细胞2型(T helper cell 2, Th2)和调节性T细胞(regulatory T cells, Treg)。近年来的研究发现,机体存在一种参与自身免疫和炎症反应的新型CD4+T细胞-辅助性T细胞17型(T helper cell 17, Th17),该细胞通过分泌产生白细胞介素17(Interleukin 17, IL-17,即IL-17A)、IL-17F、IL-21、IL-22、IL-6和肿瘤坏死因子a (Tumor Necrosis Factor a, TNF-a)等,进而发挥其集体动员、募集及活化中性粒细胞的作用。有研究报道,Th17细胞及其特征性细胞因子IL-17可能是自身免疫性心肌损伤的重要因素。如果能特异性观察M2-AA是否直接影响Thl7细胞的功能活动,则有可能进一步提供M2-AA具有调节免疫细胞功能活性的直接证据,为研究M2-AA的病理生理学意义提供新的切入点。
研究目的
(1)建立与临床患者血清中M2-AA滴度相匹配的M2-AA被动免疫大鼠模型,观察该抗体长期存在是否能够影响心脏的结构和功能,以及机体的免疫功能,以探讨M2-AA是否具有直接导致心肌损伤和调控机体免疫功能的作用。
(2)分离培养大鼠T淋巴细胞,观察M2-AA是否直接影响T淋巴细胞的功能活动,以探讨M2-AA是否可激活T淋巴细胞介导的免疫反应,并探讨M2-AA是否具有除心脏以外的作用位点。
(3)分离培养人外周血单个核细胞,刺激诱导其中初始CD4+T细胞发育分化为Th17淋巴细胞,观察M2-AA是否影响Th17淋巴细胞的功能活动。
研究方法
(1)根据大鼠M2受体细胞外第二环163-184位的氨基酸序列合成抗原肽段,建立主动免疫大鼠模型,采用SA-ELISA法监测M2-AA的产生情况。
(2)利用亲和层析法提纯免疫鼠血清中的M2-AA IgGs, SDS-PAGE凝胶电泳鉴定IgGs的纯度,采用BCA法测定蛋白含量,ELISA法鉴定所提纯的M2-AA IgGs亚类。
(3)选择血清M2-AA阴性大鼠建立M2-AA被动免疫模型,利用MS2000生物信号记录分析系统定期监测大鼠心功能的变化情况,并采集心肌组织标本,经相应处理后进行光镜和电镜观察。
(4)分离M2-AA被动免疫大鼠脾脏T淋巴细胞,进行细胞免疫荧光染色,采用流式细胞术测定CD4+/CD8+T淋巴细胞比值。
(5)急性分离培养正常大鼠脾脏T淋巴细胞,进行细胞免疫荧光染色,采用激光扫描共聚焦显微镜观察鉴定。
(6)采用CCK-8法,检测经M2-AA干预后T淋巴细胞的增殖状况,以观察M2-AA对T淋巴细胞增殖功能的影响；利用ELISA检测试剂盒,测定经M2-AA干预后的T淋巴细胞培养上清中IL-4、IL-10、IL-17、IL-21及IL-22含量。
(7)采用Ficoll-Hypaque密度梯度离心法分离人外周血单个核细胞并培养,加入诱导剂与刺激剂促进初始CD4+T淋巴细胞诱导分化为Th17淋巴细胞,进行细胞免疫荧光染色(包括细胞内因子染色),采用激光扫描共聚焦显微镜观察鉴定。
(8)采用CCK-8法,检测经M2-AA干预后Th17淋巴细胞的增殖水平,以观察M2-AA对Th17细胞增殖功能的影响；利用ELISA检测试剂盒,测定经M2-AA干预后的Th17细胞培养上清中IL-17、IL-21及IL-22含量。
研究结果
1.M2-AA的制备、提纯、定性及定量检测结果
1.1 M2抗原肽段主动免疫大鼠模型建立成功
SA-ELISA法测定结果显示：免疫后2周,与对照组相比,M2免疫组血清M2-AA的水平已明显升高,到8周时达峰值(血清M2-AA的OD值：2.93±0.11νs,s.0.21±0.02,P<0.01),之后到10周,M2-AA水平虽有下降趋势,但仍高于同期对照组(2.82±0.33νs.0.20±0.02,P<0.01)(图1-1)。本结果提示主动免疫模型建立成功。
1.2 M2-AA IgGs的提纯、定性、定量及亚类检测结果
采用亲和层析法提纯免疫鼠血清中的IgGs,得到含M2-AA的总IgGs。经SDS-PAGE凝胶电泳鉴定纯度,结果显示在55KDa和25KDa附近可见清晰的两条带,分别代表IgGs的重链和轻链(图1-2A)。
经BCA法测定纯化的IgGs蛋白浓度为2.39mg／mL。
IgGs亚类测定结果显示,所提纯的M2-AA主要为IgG2a亚类(图1-2B)。
2 M2-AA被动免疫大鼠发生心脏损伤
2.1 M2-AA被动免疫大鼠模型建立成功
M2-AA被动免疫后4周,大鼠血清M2-AA含量已明显高于对照组(OD值：0.355±0.03νs.0.103±0.04,P<0.05),之后M2-AA持续保持在一定水平,到24周时仍高于对照组(OD值：0.388±0.04νs.0.098±0.08,P<0.05)(图1-3)。结果表明M2-AA被动免疫大鼠模型建立成功。
2.2 M2-AA被动免疫大鼠的心功能下降
心功能监测中,LVSP与+dp/dtmax是反映心脏收缩功能的参数,LVEDP与-dp/dtmax反映心脏舒张功能。结果显示：被动免疫16周时,M2-AA免疫组LVEDP (KPa)明显升高
(1.53±0.91νs.同期对照组1.38±0.61,P<0.05)(图1-4B),而+dp/dtmax (KPa/sec)则出现降低(302.43±39.1νs.369.18±30.6,P<0.05)(图1-4C)。到24周时,M2-AA组LVSP (KPa)出现降低(9.06±2.20νs.13.92±2.81,P<0.05)(图1-4A),LVEDP增高(2.96±0.52νs.1.29±0.61,P<0.01),+dp/dtmax降低(213.06±40.32νs.360.32±26.81KPa/s,P<0.01),-dp/dtmax(KPa/sec)出现增加(-163.96±28.52 vs.-225.29±12.71 KPa/s, P＜0.01)(图1-4D)。
综上所述,M2-AA长期存在可导致心功能下降。
2.3 M2-AA被动免疫大鼠心重/体重比值下降
心重与体重比值(HW/BW, mg/g)测定结果显示：在免疫第16周时,M2-AA免疫组的HW/BW值明显降低(2.23±0.10νs.对照组2.48±0.11,P<0.05),到24周时降低更为明显(2.15±0.09νs.对照组2.51±0.07,P<0.01)(图1-5C)。这一结果提示,M2-AA在体内长期作用的情况下,可能发生了心肌细胞丢失现象。
2.4 M2-AA被动免疫大鼠导致心肌损伤
心脏体视学检查发现：在免疫24周时,M2-AA免疫组的心脏体积明显变大,心腔扩张(图1-5 A,B)。
光镜观察结合图像分析发现：免疫24周时,M2-AA免疫组心肌细胞形态发生明显改变,间质有淋巴细胞浸润；而对照组心肌组织形态与间质均正常(图1-6)。
观察心肌组织电镜标本发现：免疫24周时,M2-AA免疫组的心肌出现明显的线粒体肿胀、广泛空泡样变性、基质变清、线粒体嵴结构不清；心肌肌原纤维排列紊乱、明暗带缺损、部分断裂。对照组心肌超微结构显示线粒体大小、形态及分布正常；心肌肌原纤维排列整齐,肌小节结构清晰(图1-7)。
以上这些结果说明,M2-AA在体内长期存在的情况下,可导致心肌损伤。
2.5 M2-AA被动免疫后大鼠脾脏CD4+/CD8+T淋巴细胞比值增加
实验结果显示：免疫12周时,M2-AA免疫组的CD4+/CD8+比值明显增高(3.06±0.28νs.对照组1.92±0.38,P<0.05),之后呈持续增高趋势,到24周时仍明显高于对照组(3.47±0.31νs.1.82±0.33,P<0.01)(图1-8)。
这一结果提示,M2-AA在体内长期存在的情况下,可导致机体的细胞免疫功能发生紊乱。
3 M2-AA促进大鼠T淋巴细胞的增殖与分泌作用
3.1成熟T淋巴细胞的激活及鉴定
于倒置显微镜下观察可见：在分离的大鼠脾淋巴细胞中未加入ConA时,淋巴细胞呈圆形；加入ConA后刺激培养72h,细胞的体积增大,数量明显增多,形态有不规则形、长梭形、三角形等(图2-1)。
根据T淋巴细胞特征性的表面标志,采用荧光标记抗体CD3-FITC与CD4-PE进行鉴定。结果可见大部分细胞膜表面呈现绿色荧光阳性,表明是T淋巴细胞；少数细胞膜表面呈绿色与红色荧光双阳性,为辅助性T细胞(Th细胞)(图2-2)。
3.2 Con A可激活大鼠脾脏T淋巴细胞
采用CCK-8试剂盒检测淋巴细胞活性结果显示：体外培养的大鼠脾淋巴细胞中加入ConA后,细胞活性明显高于未加ConA的对照组(0.442±0.05νs.0.126±0.03,P<0.01)(图2-3)。表明Con A能够成功激活T淋巴细胞。3.3 M2-AA浓度依赖性促进大鼠T淋巴细胞增殖
实验结果显示：0.1μmol/L的M2-AA使细胞的活性水平明显升高(OD值：0.561±0.08νs.空白对照组0.439±0.05,P<0.05)(图2-4),同一浓度的氧化震颤素(选择性M2受体激动剂)也具有促T淋巴细胞增殖作用(P<0.05)。
三个浓度的M2-AA(0.01,0.1,1μmol/L)可浓度依赖性促进T淋巴细胞增殖(OD值：0.01：0.496±0.06νs.0.311±0.05,P<0.05；0.1：0.687±0.08νs.0.420±0.06,P<0.01；1：0.783±0.11νs.0.461±0.08,P<0.01)(图2-5)。同样浓度的氧化震颤素也能够浓度依赖性促进T淋巴细胞增殖。3.4 M2-AA通过M2受体介导促T淋巴细胞增殖作用
实验结果显示：T淋巴细胞悬液中同时加入M2-AA与M2受体拮抗剂Methoctraine(美索曲明)的混合液,M2-AA对T淋巴细胞的促增殖作用被抑制(图2-6)。此外,T淋巴细胞悬液中同时加入M2-AA与M2R peptide(M2受体肽段)的混合液,M2-AA对T淋巴细胞的促增殖作用也被抑制(图2-7)。这一结果表明：M2-AA是通过特异性与T淋巴细胞M2受体的细胞外第二环结合,进而实现其促T淋巴细胞增殖作用。
3.5 M2-AA能够促进T淋巴细胞分泌IL-4与IL-10
实验结果显示：0.1μmol/L与1μmol/L的M2-AA可明显增加T淋巴细胞IL-4的分泌量(图2-8)与IL-10的分泌量(图2-9),而对IL-17、IL-21、IL-22的分泌量无明显影响(表1)。
4 M2-AA促进人Th17淋巴细胞的增殖与分泌作用
4.1 Thl7淋巴细胞的形态学观察与鉴定
于倒置显微镜下观察可见：细胞液中未加任何刺激剂时,淋巴细胞呈正常的圆形；而加入一系列的刺激剂培养7天后,细胞形态发生明显改变,有不规则形、长梭形、三角形等,表明细胞已发生明显的分化(图3-1)。
由于目前尚未发现Th17淋巴细胞特征性的表面标志,故采用胞膜CD3-FITC与CD4-APC荧光抗体双染,结合胞内细胞因子IL-17-PE染色的方法进行鉴定。结果细胞膜表面呈绿色与蓝色荧光双阳性,且胞内发红色荧光者即为Thl7淋巴细胞(图3-2)。
4.2 M2-AA浓度依赖性促进Th17淋巴细胞增殖
加入诱导剂与激活剂后,Th17淋巴细胞的活性明显升高(0.398±0.12νs.对照组0.051±0.03,P<0.01)(图3-3)。表明加入诱导剂与激活剂,可使Thl7淋巴细胞活化成熟。
0.1μmol/L的M2-AA使Th17细胞活性水平明显升高(0.554±0.09νs.对照组0.419±0.05,P<0.05)(图3-4)。同一浓度的氧化震颤素也表现出相同的效应(P<0.05)。
三个浓度的M2-AA(0.01,0.1,1μmol/L)可浓度依赖性促进Th17淋巴细胞增殖(0.01：0.443±0.03νs.0.404±0.03,P<0.05；0.1：0.554±0.09νs.0.424±0.06,P<0.01；1：0.569±0.07νs.0.449±0.06,P<0.01)。同样浓度的氧化震颤素也能够浓度依赖性促进Th17淋巴细胞增殖(图3-5)。4.3 M2-AA通过M2受体介导促Th17淋巴细胞增殖作用
Thl7淋巴细胞悬液中同时加入M2-AA与M2受体拮抗剂Methoctraine(美索曲明)的混合液,M2-AA对Th17淋巴细胞的促增殖作用被抑制(图3-6)。此外,Th17淋巴细胞悬液中同时加入M2-AA与M2R peptide(M2受体肽段)的混合液,M2-AA对Th17淋巴细胞的促增殖作用也被抑制(图3-7)。这一结果表明：M2-AA是通过特异性与Th17淋巴细胞M2受体的细胞外第二环结合,进而实现其促Th17淋巴细胞增殖作用。4.4 M2-AA能够促进Th17细胞分泌IL-17、IL-21与IL-22
0.1μmol/L的M2-AA可明显增加Thl7细胞IL-17的分泌量(0.049±0.01νs.0.043±0.01,P<0.05)(图3-8),及IL-21的分泌量(0.048±0.01 vs.0.042±0.01,P<0.05)(图3-9),并且1μmol/L的M2-AA这些作用更为明显(P<0.01)。此外,1μmol/L的M2-AA可明显增加Th17细胞IL-22的分泌量(0.053±0.01νs.0.044±0.01,P<0.05)(图3-10)。同样,0.1μmol/L与1μmol/L的氧化震颤素也可增加Th17细胞对这三种细胞因子的分泌量。结论(1)M2-AA以和临床患者血清滴度相匹配的浓度在体内长期存在的情况下,可引发心肌损
伤和机体细胞免疫功能紊乱。(2)在一定范围内,M2-AA可浓度依赖性促进体外培养的T淋巴细胞增殖,并且增殖的T
淋巴细胞分泌功能增强。表明除心肌细胞以外,T淋巴细胞的M2受体也是M2-AA的
作用靶点。(3)在一定范围内,M2-AA可剂量依赖性促进体外培养的Th17淋巴细胞增殖,并且增殖
的Th17淋巴细胞分泌功能增强。
Background
Since the nineties of last century, many researchers found that there were autoantibodies against the second extracellular loop of M2-muscarinic acetylcholine receptor (M2-AA) in the serum of patients with cardiovascular disease in high titer. Further functional studies found that the M2-AA played a role of negative chronotropic effect on the myocardial cells in vitro, which was similar to M2 receptor agonist carbachol, but the stimulation is not easy to desensitization. Therefore, we speculate that maybe the M2-AA is able to interfere with the nomal function of heart according to activating M2 receptor in the long-term, which result in the pathological injuries to the heart.
We have immunized rats with the synthesis peptides against the second extracellular loop of the M2 receptor as antigens for long time, then we observed:with the increase of M2-AA titer and extend of interaction time, myocardial remodeling occurred gradually, in the end, the the pathological and functional changes which was similar to of dilated cardiomyopathy appeared. During the immunization time, the degeneration of vacuolar presented in mitochondria of myocardial cells, with the loss of myocardial cells in focus, and filtration of lymphocytes. These results suggested that the M2-AA may be involved in autoimmune-mediated pathogenesis of heart disease.However, the above-mentioned study is not sufficient to conclude that M2-AA can result in cardiac injuries directly. Because the model was active immunization model, in one hand, the else factors except M2-AA can not be excluded in the model, for instance, whether the antigen is involved in the occurance of cardiac injuries; on the other hand, the titer of M2-AA produced by active immunization model highly more than the serum of the clinical patients. Thus, it is difficult to explain the clinical cases by active immunization model. Consequently, establishment of passive immunization model of animals whose concentration of M2-AA similar to the clicical situations is the essential and basic experiment method of the further study about the pathophysiology significance of M2-AA.
In the active immunization model above, the infiltration of a large number of lymphocytes in the damaged myocardial tissue was also an important pathological phenomenon, hinting that a certain factors activated the immune system of an organism. As we all know, T lymphocytes are the responsible for the immunologic response, which have multiple biological functions of killing the target cells directly, assisting or inhibiting the antibodies produce by B cells, specific response of antigens and promoting mitogen, and production of cytokines and such on. It is reported that there are M2 receptors in the T lymphocytes of human and murine, and the parasympathetic neurotransmitter ACh plays a role in immune regulation. In view of M2-AA is similar to agonist-like activation to M2 receptors, so we speculated that M2-AA in the circulating blood may also activate M2 receptors of T lymphocytes, thereby affecting the activity of T lymphocytes and immune function. However, whether M2-AA is directly involved in activation of T lymphocytes needs to be further identified. If it is, the forms of participation need to be studied.
In the specific immune response of body, CD4+T lymphocytes are the main effective T cells involved in all stages of the immune response, and play a key role in immune regulation. According to the traditional view, CD4+T lymphocytes were divided into type 1 T helper cells (T helper cell 1, Th1), type 2 T helper cells (T helper cell 2, Th2) and regulatory T cells (regulatory T cells, Treg). In recent years, researchers found that there is a new CD4+T lymphocytes intype17 T helper cells (T helper cell 17, Th17), which involved in body's own immune and inflammatory responses. Th17 lymphocytes can secret interleukin-17 (IL-17, namely IL-17A), IL-17F, IL-21, IL-22, IL-6, TNF-a (Tumor Necrosis Factorα,) and so on. These cytokines play the role of collective mobilization, collection and activated neutrophils. Some studies reported that Th17 cells and their characteristic cytokines IL-17 may be an important autoimmune factors in myocardial injury. If we can confirm whether M2-AA directly affect the functional activity of Th17 cells specifically, may be it will provide direct evidence that M2-AA is capable of regulating immune cell function and activity, which supply a new starting point of further study of the physiological significance to M2-AA.
Objective
(1) The passive immunization rat models were establishment by injection of M2-AA to observe whether long-term presence of M2-AA can lead to changes of cardiac structure and function, as well as in immune function of body, in order to explore whether the M2-AA has a direct role in leading to myocardial injury and regulation of immune function.
(2) Isolation and culture of rat T lymphocytes to observe whether the M2-AA could affect the proliferation and secretion of T lymphocytes, in order to investigate whether the M2-AA can activate T-lymphocyte-mediated immune response, and explore other role sites of M2-AA except to the heart.
(3) Isolation and culture of human peripheral blood mononuclear cells, the native CD4+T cells were induced by the inducers and activators to differentiate to Th17 lymphocytes, in order to observe the influence of M2-AA on functional activites of Th17 lymphocytes.
Methods
(1) The rats model of active immunization are established with synthetic antigenic peptide according to 163-184 amino acid sequence of the second extracellular loop of M2 receptor, and SA-ELISA method is used for monitoring the production of M2-AA.
(2) Affinity chromatography is used to purify M2-AA IgGs in the serum of the immune rats. SDS-PAGE gel electrophoresis is used for determine the purity of IgGs, and BCA method is used for measuring the content of proteins. ELISA method is used for identifying the purified M2-AA IgGs subsets.
(3) Passive immunization rat models of M2-AA were established by the choosing the serum M2-AA negative animal. The MS2000 Biological Signal Recording and Analysis System were used to monitor the cardiac function in a regular time, and myocardial tissue samples were collected and treated to observe under the light and electron microscope.
(4) The T lymphocytes from spleen in rats passive immunization M2-AA were isolated. Cell staining by immunofluorescence was used, and CD4+/CD8+T-lymphocyte ratio is determined by flow cytometry.
(5) The T lymphocytes from spleen of normal rats were acutely isolated and cultured, then the cell staining by immunofluorescence was used and the T lymphocytes were identified by laser scanning confocal microscope.
(6) CCK-8 method was used to detect the activity levels of T-lymphocyte after the accession of M2-AA, in order to determinate the impact of M2-AA on the proliferation function of T-lymphocyte. ELISA kits wer used to detect the levels of IL-4, IL-10, IL-17, IL-21 and IL-22 in the supernatant of cultured T-lymphocyte after intervention by M2-AA.
(7) Ficoll-Hypaque density gradient centrifugation was used to isolate and culture the peripheral blood mononuclear cells in human. Th17 cells were promoted to differentiate and maturate by adding inducing agents and stimulants, and cell staining by immunofluorescence (including intracellular cytokine staining) was used, in order to identify them by laser scanning confocal microscope.
(8) CCK-8 method was used to detect activity level of Th17 cells after intervention by M2-AA, to observe the impact on the proliferation function of Th17 cells by M2-AA; ELISA kits were used to detect the levels of IL-17, IL-21 and IL-22 in the supernatants of cultured Th17 cell after intervention by M2-AA.
Results
1. Preparation, purification, qualitation and quantitation of M2-AA
1.1 The active immunized rat models were set up successfully
The results determined by SA-ELISA showed that 2 weeks after immunization, the level of M2-AA in the sera of M2 immunized group has already raised and reached the peak in 8 weeks (OD Values:2.93±0.11 vs.0.21±0.02, P＜0.01). Then to 10 weeks, the levels of M2-AA decreased, but it was still higher than that before initial immunization (P＜0.01) and the control group (P＜0.01). (Fig.1-1). The result suggests that the active immunized rat models were set up successfully.
1.2 Purification, quantitative qualitative and detection the subtype of M2-AA IgGs
IgGs in the sera of immunized group were purified by affinity chromatography, so the total IgGs including M2-AA were obtained. The specificity of purified IgGs was determined by the SDS-PAGE. The results showed that two straps of 55KD and 25KDa appeared which represented one heavy chain and one light chain, respectively (Fig.l-2A).
The concentration of purified IgGs was 2.39 mg/mL, which was determined by BCA kits.
The subtype of purified M2-AA IgGs was mainly IgG2a (Fig.l-2B).
2. The heart injury appeared in the M2-AA passive immunized rat models
2.1 The passive immunized rat models were established successfully
4 weeks after passive immunization, the sera level of M2-AA in the immunized group was significantly higher than that in the control group (0.355±0.03 vs.0.103±0.04, P<0.05). Then the sera level of M2-AA persisted at stable level. It was still higher than that in control group in 24 weeks after immunization (0.388±0.04 vs.0.098±0.08, P＜0.01) (Fig.1-3). The result suggests the passive immunized rat models were set up successfully.
2.2 The cardiac function decreased in the passive immunized rat
16 weeks after passive immunization, the LVEDP in the M2-AA group has been higher than that in the control group (1.53±0.91 vs.1.38±0.61, P<0.05) (Fig.l-4B), while the+dp/dtmax was significantly lower than the control group (302.43±39.1 vs.369.18±30.6, P＜0.05) (Fig.l-4C). In 24 weeks in the M2-AA immune rats, the LVSP was lower than that in the control group (9.06±2.20 vs.13.92±2.81, P<0.05), but the LVEDP was higher than the corresponding control group (2.96±0.52 vs.1.29±0.61, P<0.01),+dp/dtmax decreased (213.06±40.32 vs.360.32±26.81, P<0.01) (Fig.l-4A), and the-dp/dtmax increased(-163.96±28.52 vs.-225.29±12.71, P<0.01) (Fig.l-4B, D), these results indicate decline in cardiac function.
2.3 The ratios of heart weight to body weight (HW/BW) decreased in the passive immunized rats
16 weeks after passive immunization, the ratio of heart weight to body weight (HW/BW) was significantly lower than that in the control group (2.23±0.10 vs.2.48±0.11, P＜0.05), and it decreased continuously until the experiment was ended up (Fig.1-5C).
2.4 Myocardial injury induced by passive immunization rats with M2-AA
Heart stereological examination showed 24 weeks after immunization with M2-AA, the heart size became larger and the heart chamber expanded (Fig.l-5A, B). While there were no significant changes in the heart at other time points of immunization.
HE staining of myocardial tissue showed:24 weeks after immunization, there were immune inflammatory cells infiltration in the myocardial cells of M2-AA group, and the emergence of infiltration with inflammatory cells, while the control group without such changes in myocardial tissue (Fig.1-6).
Electron microscope displayed marked myocardial fibrosis, deposition with collagen in sarcoplasmic reticulum, and swelling and degeneration in mitochondria, while the control group does not appear similar to myocardial change(Fig.l-7).
These results suggest that long-standing of M2-AA in vivo can lead to myocardial injury.
2.5 The ratios of CD4+/CD8+ T-lymphocytes from spleen cells increased in passive immunization of rats with M2-AA
Before 12 weeks after immunization, there was no significant difference in ratios of CD4+/CD8+ T-lymphocytes in the M2-AA immune group and the control group. It was significantly higher in the M2-AA group at 12th week (3.06±0.28 vs.1.92±0.38, P＜0.05), and it continuous increase to 24 weeks with more notable difference (3.47±0.31 vs.1.82±0.33, P＜0.01) (Fig.1-8).
This result indicates that long-standing of M2-AA in vivo leads to cellular immune function disturbance in the body.
3. M2-AA accelerated proliferation and secretion of T lymphocytes
3.1 The activation and identification of mature T lymphocytes
The T lymphocytes were round or oval-shaped without ConA; when were stimulated and cultured for 72h by, the cell volume augmented, with the number increased significantly, and the pattern were irregular-shaped, long spindle or triangular, etc(Fig.2-1).
The T lymphocytes were identified by immunofluorescence, according to the characteristic surface markers of T lymphocytes. The fluorescently labeled antibodies of CD3-FITC and CD4-PE were used. The result showed most of the cell surface display of green fluorescent-positive, indicating that T lymphocytes; a small number of cell surface was green and red fluorescence double positive, these were the helper T cells (Th cells) (Fig.2-2).
3.2 T lymphocytes from rat spleen could be activated by Con A
After adding ConA to the cultured rat spleen lymphocytes, the cell activity was significantly higher than that of the control group (OD value,0.442±0.05 vs.0.126±0.03, P＜0.01) (Fig.2-3), which indicated that Con A could promote T lymphocytes proliferation.
3.3 M2-AA promoted proliferation of T lymphocytes in a dose-dependent manner
O.1μmol/L of the M2-AA elevated the cell activity significantly (OD values,0.561±0.08 vs. 0.439±0.05, P<0.05), the Oxotremorine also showed the similar effect in the same concentration (P＜0.05) (Fig.2-4).
Three concentrations of M2-AA (0.01,0.1, 1μmol/L) could promote T lymphocyte proliferation in a concentration-dependent manner (OD values:0.01:0.496±0.06 vs.0.311±0.05, P<0.05; 0.1:0.687±0.08 vs.0.420±0.06, P＜00.01; 1:0.783±0.11 vs.0.461±0.08, P＜0.01). The same concentration of Oxotremorine also promoted T lymphocytes proliferation in a dose-dependent (Fig.2-5).
3.4 M2-AA promoted T-lymphocyte proliferation through the M2 receptor
The effect of M2-AA on the proliferation of T lymphocytes could be inhibited by the M2 receptor antagonist Methoctraine (Fig.2-6). In addition, the effect of M2-AA could also be inhibited by M2R peptides (Fig.2-7). The results indicate that the M2-AA promoted T-lymphocyte proliferation through the M2 receptor. 3.5 M2-AA increased the secretion of IL-4 and IL-10 by T-lymphocyte
0.1μmol/L and 1μmol/L of the M2-AA were able to increase the secretion of IL-4 (Fig.2-8) and IL-10 (Fig.2-9) by T-lymphocyte, while M2-AA had no effect on the secretion of IL-17, IL-21or IL-22(Tab.1).
4. M2-AA promoted the proliferation and secretion of Th17 lymphocytes
4.1 The morphology and identification of Th17 lymphocytes
The lymphocytes showed a normal round or oval without any stimulant. When added stimulants for 7 days, the cell morphology changed significantly, they were in irregular shapes, spindle-cell type, triangular, etc. Which indicated that the Thl7 lymphocytes have been differentiated (Fig.3-1).
As there are no characteristic surface markers of Th17 cells so far, we used the membrane CD3-FIT.C and CD4-APC fluorescence antibody staining, combined with intracellular cytokine IL-17-PE staining methods to identify the Th17 lymphocytes. The surface of Th17 lymphocytes cell were green and blue fluorescent double-positive, moreover, the red fluorescence was detected in intracellular (Fig.3-2).
4.2 M2-AA induced a dose-dependent proliferation of Th17 lymphocytes
After added the inducers and activators, the activities of Th17 lymphocytes increased significantly (0.398±0.12 vs.0.051±0.03, P＜0.01), which suggested that add inducers and activators could promote the mature of Th17 lymphocytes (Fig.3-3).
0.1μmol/L of the M2-AA increased the activity of Th17 lymphocytes (0.554±0.09 vs. 0.419±0.05, P<0.05). The same concentration of Oxotremorine also exhibited the same effects (P＜0.05) (Fig.3-4).
Three concentrations of M2-AA (0.01,0.1, 1μmol/L) promoted Th17 lymphocytes proliferation in a concentration-dependent manner (0.01:0.443±0.03 vs.0.404±0.03, P<0.05; 0.1: 0.554±0.09 vs.0.424±0.06, P<0.01; 1:0.569±0.07 vs.0.449±0.06, P＜0.01). The same concentration of Oxotremorine also promoted Th17 lymphocytes proliferation by a concentration-dependent manner (Fig.3-5).
4.3 M2-AA promoted Th17 lymphocyte proliferation through the M2 receptor
The effect of M2-AA on the proliferation of Th17 lymphocytes could be inhibited by the M2 receptor antagonist Methoctraine (0.449±0.03 vs.0.439±0.05, P>0.05) (Fig.3-6). In addition, the effect of M2-AA could also be inhibited by M2R peptides (0.437±0.04 vs.0.439±0.05, P>0.05) (Fig.3-7). These results indicate that the M2-AA promoted Th17 lymphocytes proliferation through the M2 receptor.
4.4 M2-AA promoted Th17 cell secretion of IL-17, IL-21 and IL-22
0.1μmol/L of the M2-AA increased the secretion of IL-17 (0.049±0.01 vs.0.043±0.01, P＜0.05) and IL-21 (0.048±0.01 vs.0.042±0.01, P＜0.05) by the Thl7 lymphocytes (Fig.3-8,3-9), the effects of 1μmol/L M2-AA were more obvious (P＜0.01). In addition, 1μmol/L of the M2-AA increased the secretion of IL-22 (0.053±0.01 vs.0.044±0.01, P＜0.05) (Fig.3-10). Similarly, 0.1μmol/L and 1μmol/L Oxotremorine also increased the secretion of the three kinds of cytokines.
Conclusions
(1) The M2-AA can lead to myocardial injury and cellular immune function disturbance in the condition of long-standing and match the concentration of the clinical serum titer.
(2) Within a certain range, M2-AA promotes T-lymphocyte proliferation and enhances the secretion of cytokines in a concentration-dependent manner in vitro. In addition to the cardiomyocyte, the T lymphocytes are also the role of M2-AA target.
(3) Within a certain range, M2-AA promoted the proliferation of Th17 lymphocytes in dose-dependent manner in vitro, and increased the function of secretion inflammatory cytokines.
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